Ana-Sunčana Smith

Force-induced growth of adhesion domains is controlled by receptor mobility

In living cells, adhesion structures have the astonishing ability to grow and strengthen under force. Despite the rising evidence of the importance of this phenomenon, little is known about the underlying mechanism. Here, we show that force-induced adhesion-strengthening can occur purely because of the thermodynamic response to the elastic deformation of the membrane, even in the absence of the actively regulated cytoskeleton of the cell, which was hitherto deemed necessary. We impose pN-forces on two fluid membranes, locally pre-adhered by RGD-integrin binding. One of the binding partners is always mobile whereas the mobility of the other can be switched on or off. Immediate passive strengthening of adhesion structures occurs in both cases. When both binding partners are mobile, strengthening is aided by lateral movement of intact bonds as a transient response to force-induced membrane-deformation. By extending our microinterferometric technique to the suboptical regime, we show that the adhesion, as well as the resistance to force-induced de-adhesion, is greatly enhanced when both, rather than only one, of the binding partners are mobile. We formulate a theory that explains our observations by linking the macroscopic shape deformation with the microscopic formation of bonds, which further elucidates the importance of receptor mobility. We propose this fast passive response to be the first-recognition that triggers signaling events leading to mechanosensing in living cells.

Physics of cell adhesion: some lessons from cell-mimetic systems

Cell adhesion is a paradigm of the ubiquitous interplay of cell signalling, modulation of material properties and biological functions of cells. It is controlled by competition of short range attractive forces, medium range repellant forces and the elastic stresses associated with local and global deformation of the composite cell envelopes. We review the basic physical rules governing the physics of cell adhesion learned by studying cell-mimetic systems and demonstrate the importance of these rules in the context of cellular systems. We review how adhesion induced micro-domains couple to the intracellular actin and microtubule networks allowing cells to generate strong forces with a minimum of attractive cell adhesion molecules (CAMs) and to manipulate other cells through filopodia over micrometer distances. The adhesion strength can be adapted to external force fluctuations within seconds by varying the density of attractive and repellant CAMs through exocytosis and endocytosis or protease-mediated dismantling of the CAM–cytoskeleton link. Adhesion domains form local end global biochemical reaction centres enabling the control of enzymes. Actin–microtubule crosstalk at adhesion foci facilitates the mechanical stabilization of polarized cell shapes. Axon growth in tissue is guided by attractive and repulsive clues controlled by antagonistic signalling pathways.

Progress in Mimetic Studies of Cell Adhesion and the Mechanosensing

Vesicle-substrate adhesion has been studied for over two decades with the motivation to understand and mimic cell adhesion. In recent years, with progress in theoretical modelling, the development of experimental techniques, and improved data-analysis procedures, considerable advances have been made in the understanding of the adhesion process. It is this progress which constitutes the focus of this review.

Vesicles as a model for controlled (de-)adhesion of cells: a thermodynamic approach

We review the specific adhesion between ligand-containing vesicles and receptor-functionalized substrates as an established model system used to study the cell recognition process and its control mechanisms. In order to provide better understanding of the underlying physics and to allow for quantitative exploitation of this system, we develop a simple theoretical framework that accounts for the equilibrium state of adhesion and successfully merges the macroscopic and microscopic aspects of the problem. Several mechanisms that are used to control adhesion or induce de-adhesion are studied on the same level of theory. Specifically, the repelling properties of adhesive molecules, the role of repelling molecules, the action of antagonists for a specific binder as well as the influence of an externally applied force are addressed independently within the same formalism.

Inter-membrane adhesion mediated by mobile linkers: Effect of receptor shortage

Giant unilamellar vesicles (GUVs) adhering to supported bilayers were used as a model system to mimic ligand–receptor mediated cell-cell adhesion. We present the effect of varying the concentration of receptors (neutravidin on the bilayer) and ligands (biotin on the vesicle) on GUV adhesion and the organization of receptors in the adhesion zone. At high concentrations of both ligands and receptors, the adhesion is strong, all the available membrane is adhered and receptors are accumulated under the adhered membrane up to the geometrical limit of close packing. At low concentrations of receptors (<0.5%), and an arbitrary concentration of ligands (≥0.1%), adhesion does not proceed to completion: the membrane is only partially bound and parts of it still fluctuate. The receptors tend to accumulate under the adhered membrane but the filling is partial. Receptors get jammed and form clusters with fractal like shapes along the rim of the adhered vesicle in such a way that the annular cluster prevents further filling of the adhesion disc. We characterize the filling in terms of a compaction factor and the final concentration. Interestingly, the closing of the ring of jammed clusters switches the interior of the adhesion disc from one thermodynamic ensemble to another. In the new ensemble the receptors sealed within the adhesion disc are mobile but their number is fixed. Under such conditions, the usually strong neutravidin/biotin bond is weak. The incomplete adhesion state can be attributed to a combination of the effects of diffusion, jamming and the competition of enthalpy and entropy on bond formation. The formation of jammed receptor clusters reported here represents a new mechanism that influences membrane adhesion.

Measuring fast stochastic displacements of bio-membranes with dynamic optical displacement spectroscopy.

Stochastic displacements or fluctuations of biological membranes are increasingly recognized as an important aspect of many physiological processes, but hitherto their precise quantification in living cells was limited due to a lack of tools to accurately record them. Here we introduce a novel technique—dynamic optical displacement spectroscopy (DODS), to measure stochastic displacements of membranes with unprecedented combined spatiotemporal resolution of 20 nm and 10 μs. The technique was validated by measuring bending fluctuations of model membranes. DODS was then used to explore the fluctuations in human red blood cells, which showed an ATP-induced enhancement of non-Gaussian behaviour. Plasma membrane fluctuations of human macrophages were quantified to this accuracy for the first time. Stimulation with a cytokine enhanced non-Gaussian contributions to these fluctuations. Simplicity of implementation, and high accuracy make DODS a promising tool for comprehensive understanding of stochastic membrane processes.

Switching from Ultraweak to Strong Adhesion

Nature switches from weak to strong adhesion at the cellular level to spectacular effect – for example, for incredibly sensitive recognition and decisive action during immune response. [ 1 ] If realized in an artifi cial system, such a switching could one day be harnessed as a powerful tool to manipulate weakly interacting objects. The fi rst step towards realizing such a system involves understanding how to create and detect ultraweak adhesion and how to then switch-on a strong interaction. So far, in the context of model membranes, weak adhesion has been achieved only with a ligand-receptor of intrinsically low binding affi nity. [ 2 ] Whatever, the intrinsic strength of the bonds, so far they were usually found to be arranged in compact stable domains. [ 3 ] Here, we present experiments and simulations that indicate how to create and detect ultraweak adhesion in the context of fl uid two dimensional membranes interacting via specifi c ligand/receptor bonds. Thus, specifi c adhesion is mediated by transient domains consisting of sparsely distributed bonds. Amazingly, we demonstrate that the avidin/biotin pair – famous for forming the strongest receptor/ligand bond known in nature, mediates ultraweak adhesion under suitable circumstances. This choice of binders allows us to switch on strong binding once sensitive detection is achieved – without resorting to a second binding pair – something not possible with intrinsically weak binders. However, this goal necessitates an appropriate design strategy elaborated below. The in vitro system consists of two membranes: a solid supported lipid bilayer (SLB) and the freely fl uctuating membrane of a giant unillamelar vesicle (GUV). A GUV is a two

Complementary Molecular Dynamics and X-ray Reflectivity Study of an Imidazolium-Based Ionic Liquid at a Neutral Sapphire Interface.

Understanding the molecular-level behavior of ionic liquids (ILs) at IL-solid interfaces is of fundamental importance with respect to their application in, for example, electrochemical systems and electronic devices. Using a model system, consisting of an imidazolium-based IL ([C2Mim][NTf2]) in contact with a sapphire substrate, we have approached this problem using a complementary combination of high-resolution X-ray reflectivity measurements and atomistic molecular dynamics (MD) simulations. Our strategy enabled us to compare experimental and theoretically calculated reflectivities in a direct manner, thereby critically assessing the applicability of several force-field variants. On the other hand, using the best-matching MD description, we are able to describe the nature of the model IL-solid interface in appreciable detail. More specifically, we find that characteristic interactions between the surface hydroxyl groups and donor and acceptor sites on the IL constituents have a dominant role in inducing a multidimensional layering profile of the cations and anions.

Inferring spatial organization of bonds within adhesion clusters by exploiting fluctuations of soft interfaces

Detecting the organization of bonds within adhesion domains connecting two interacting membranes is, at present, extremely challenging. Herein we present a technique, based on Reflection Interference Contrast Microscopy, which uses spontaneous thermal fluctuations of a soft interface as a tool to identify the organization of specific ligand-receptor bonds. The key is a time-resolved analysis of micro-interferometric data that systematically quantifies fluctuations and enables the detection of their suppression due to the formation of bonds, which, in turn, allows the identification of the bond organization without the use of fluorescent labelling. The identification of a new type of bond organization characterized by sparsely distributed bonds, as well as detection of pinning centres of nanometric size is presented.

The Protonation States of the Active-Site Histidines in (6–4) Photolyase

The active sites of the (6-4) photolyases contain two conserved histidine residues, which, in the Drosophila melanogaster enzyme, correspond to His365 and His369. While there are nine combinations in which the three possible protonation states of the two histidines (with protons on Nδ (HID), Nε (HIE), or both Nδ and Nε (HIP)) can be paired, there is presently no consensus as to which of these states is present, let alone mechanistically relevant. EPR hyperfine couplings for selected protons of the FADH(•) radical have previously been used to address this issue. Our QM/MM calculations show, however, that the experimental couplings are equally well reproduced by each of the nine combinations. Since the EPR results seemingly cannot be used to unequivocally assign the protonation states, the pKa values of the two histidines were calculated using the popular PROPKA, H++, and APBS approaches, in various environments and for several lesions. These techniques consistently indicate that, at pH = 7, both His365 and His369 should be neutral, although His369 is found to be more prone to becoming protonated. In a comparative approach, a series of molecular dynamics simulations was performed with all nine combinations, employing various reference crystal structures and different oxidation states of the FAD cofactor. The overall result of this approach is in agreement with our pKa results. Consequently, although the introduction of the reduced cofactor results in an increased stability for selected protonated states, particularly the His365═HID and His369═HIP combination, the neutral combination His365═HID and His365═HIE stands out as the most relevant state for the activity of the enzyme.